Anthracnose caused by Colletotrichum graminicola (Ces.) G.W.Wils is a significant disease of maize (Zea mays) worldwide. To obtain an efficient biocontrol strain and elucidate its mechanisms, 103 bacterial isolates were obtained from soil samples collected in the Tianshan Mountains, Xinjiang, China. Among these, Bacillus atrophaeus F4’s fermentation broth had the highest efficacy in controlling maize anthracnose, reaching 79.78%. To further investigate biocontrol mechanisms of F4 strain, its complete genome was sequenced, assembled, and annotated. Lipopeptides extracted from the fermentation broth of F4 were found to strongly inhibit the growth of hyphae and the germination of conidia in the pathogen. Microscopic and biochemical analyses indicated that the lipopeptide extract inhibited chitin synthesis and disrupted the integrity of the cell wall and membrane, thereby exerting antifungal effects. Further MALDI-TOF MS analysis identified antimicrobial compounds, including surfactin, iturin, and fengycin B, in the lipopeptide extract. Furthermore, plate antagonistic test showed that F4 strain exhibited broad-spectrum antagonistic activity against multiple plant pathogenic fungi. F4 strain also displayed motility, biofilm-forming capacity, and the ability to produce extracellular enzymes such as proteases and amylases, which are associated with biocontrol activity. These findings suggest the significant potential of B. atrophaeus F4 as a biocontrol agent against maize anthracnose.